Autoreactive T cells that are capable of inducing disease exist in normal adult animals, but are maintained in a dormant or inactive state due to the suppressive functions of regulatory T cells (Treg). We have demonstrated that the regulatory T cells can be easily identified in normal lymphoid tissues by expression of CD4, the interleukin-2 receptor alpha chain (CD25), and the transcription factor, FoxP3. Transfer of CD4+CD25-Foxp3- T cells to immunoincompetent mice results in the development of autoimmune disease that can be prevented by co-transfer of CD4+CD25+Foxp3+ T cells. The major goals of this project are to define the function and mechanism of action of Treg cells in vivo. We have used both polyclonal Treg and Treg that have been induced in vitro by stimulation of naive T cells in the presence of TGF-beta. Some studies have suggested that iTreg lack regulatory function even though they express Foxp3 and that they rapidly lose Foxp3 expression following transfer in vivo. As adoptive transfer of Treg into neonatal Scurfy mice prevents the development of disease, we tested whether iTreg would also be effective in rescuing Scurfy mice. We found that polyclonal iTreg generated from peripheral CD4+Foxp3- T cells could completely suppress all the pathologic manifestations of the severe autoimmune disease that develops in Scurfy mice in both lymphoid sites and in tissues. Weekly injections of iTreg prolonged the life of the scurfy mice for more than 35 days. Thus, the TCR repertoire of the iTreg generated from normal adult mice appears to be sufficient to prevent autoimmune disease in the scurfy mouse. The iTreg maintained high levels of Foxp3 expression in the inflammatory environment, whereas the same cells when transferred to normal mice lost expression of Foxp3, suggesting that certain factors such as proinflammatory cytokines in the sick Scurfy mouse might act as survival or expansion signals for the iTreg. Taken together, these results demonstrate the potency and stability of the iTreg in curing the most severe autoimmune disease. We have demonstrated that naive H+/K+ ATPase-specific CD4+CD8-Foxp3- thymocytes from TCR transgenic mice can be easily expanded and converted to FoxP3+ T cells when activated in the presence of TGF-beta. Most importantly, these autoantigen-specific iTregs were able to potently inhibit the development of AIG. iTregs prevented the priming and expansion of the effector T cells very early after cell transfer and appeared to decrease the stimulatory capacity of dendritic cells (DC) presenting endogenous autoantigen rather than by competing with the effectors for antigen or by acting directly with the effector cells to prevent their interaction with the DC. Thus, iTregs stop the autoimmune reaction before it even starts. Most studies of Treg function in models of organ-specific autoimmune diseases have focused on prevention of disease induced by nave T cells rather than on treatment of ongoing disease or prevention of disease induced by fully differentiated effector T cells. We have compared the capacity of fully differentiated Th1, Th2 and Th17 cells derived from the TxA23 mouse to induce AIG upon transfer to nu/nu recipients and determined the susceptibility of these fully differentiated effector T cell populations to control by transfer of polyclonal Treg. We could readily induce differentiation of thymocytes from the TxA23 mouse to Th1, Th2, and Th17 cell lines. After transfer, all 3 effector T cell populations preferentially accumulated in the gastric LN and the gastric mucosa and elicited destructive AIG with distinctive pathologic profiles. Th17-mediated disease seemed to be the most aggressive type and was accompanied by the highest number of infiltrating polymorphonuclear leukocytes including a very high percentage of eosinophils. Polyclonal Treg could suppress the capacity of Th1 cells, moderately suppress Th2 cells, but had little effect on Th17-induced AIG. In contrast to our studies with nave T cells, significant inhibition of effector cell expansion was seen when polyclonal Treg were co-transferred with Th1 and Th2 effectors and the inhibition of expansion was an excellent correlate of protection from destructive AIG. One reason for this result is that the effector populations expressed only low levels of CD62L and could not enter the gastric lymph node, while the Treg express high levels of CD62L and could easily be detected in the gastric lymph node immediately after transfer. A small percentage of the polyclonal Treg population expressing receptors for antigens derived from the target organ may have been retained in the node, expanded, and then exerted inhibitory effects on DC resulting in inhibition of the expansion of the susceptible effector populations upon their arrival in the node. T effector cells isolated from protected animals were not anergic and were fully competent to produce effector cytokines ex vivo. These findings are most consistent with a model where Tregs in vivo are not shutting off effector cytokine production by T cells directly, but are regulating their expansion by acting on DC. Unlike Th1, Th2 and naive CD4+ T cells, Th17 could not be suppressed in vivo by co-transfer of polyclonal regulatory T cells (nTreg). Even at the high Treg to effector ratio of 20:1 co-transfer of polyclonal Treg did not prevent Th17-mediated disease. This result led us to question whether differentiated Th17 cells are intrinsically unresponsive to suppression by Treg. In order to test this hypothesis, we compared different types of Treg for their suppressive potential in Th17-mediated autoimmune disease, including polyclonal and antigen-specific TGF-beta-induced Treg (iTR). We found that in line with previous results, polyclonal nTregs did not suppress Th17 mediated AIG in vivo and also TGF-beta-induced polyclonal iTR had no effect. In contrast, TxA23 antigen-specific iTR effectively suppressed Th17-mediated AIG. Co-transfer of TGF-beta-induced antigen-specific iTR completely prevented tissue destruction in the gastric mucosa. Lower pathology scores were accompanied by reduced numbers of transgenic effector T cells in the draining lymph node. Furthermore, in a therapeutic approach, TxA23 transgenic iTR suppressed Th17-mediated AIG when injected one week after transfer of the Th17 effectors. Thus, TGF-beta--induced antigen-specific iTR are capable of both preventing and treating autoimmune gastritis induced by fully differentiated Th17 effector cells.